VL4: Some molecular methods in infection biology

Question 1

What are some typical questions in infection biology?

Answer 1

-Whar organisms is causing the infection?
-How does it cause the infection?
What are the relevent virulence factors?

Question 2

Define pathogenicity, virulence and virulence factor

Answer 2

Pathogenicity: ability to cause disease
Virulence: degree of pathogenicity
Virulence factor: cellular factors contributing to virulence

Question 3

WHich factors are important in vitro, in vivo, virulence?

Answer 3

Essential factors are not always the same in vivo and in vitro, in vivo. virulence factors are not important in vitro, so it is hard to study them in vitro, not all factors that are essential in vivo are essential in vitro (some metabolic pathways enzymes and virulence factors ect…) not all virulence factors are essential in vivo ( QS, secretion systems …

Question 4

Name methods to study cell biology, DNA, RNA, prot

Answer 4

Cell biology:

• Cell growth
• Microscopy

DNA:

• PCR
• Genome sequencing
• Bioinformatics

RNA:
RT-PCR
cDNA microarrays

Protein
-WEster blots
Proteomics
-Transfections
-Chemical genetics

Question 5

What is the advantage of histology?

Answer 5

You can see changes in healthy/unhealthy tissues

bacteria, fibrin deposition, inflammatory cell infiltrate

Question 6

Explain the prinziple of blotting

Answer 6

DNA/RNA/Protein sample separated on gel –> transfer to paper, visualize specific bands of interest with probes

Question 7

Type of blots for which molecule?

Answer 7

Southern blot –> DNA
Northern blot: RNA
Western blot Protein

Question 8

Explain the principle of PCR?

Answer 8

Denaturation (95°C), Annealing (50-60°C), Extension (68-72°C)

Question 9

Which ingredients are neede for PCR?

Answer 9

DNA (template), Primers (forward, reverse), DNA polymerase, Salt (MgCl2), dNTPs (ATGC)

Question 10

What is qPCR used for?

Answer 10

quantify DNA or RNA

• how many copies of a DNA sequence in genome?
• how many RNA transcripts of a gene?
• How many copies of a dna or rna in a sample?

quantification usually relative to control standard, but also absolute quantification

Question 11

What are Ct value and threshold?

Answer 11

Ct= number of cycles required to reach threshold
threshold is amount of fluorescence required to detect sample/fluorescence above background

the lower the ct the more abundant the inital target

Question 12

What type of primers can be used for qRT-PCR

Answer 12

specific
random hexamer
polyT
cDNA used a template for PCR

Question 13

How does the TaqMan method foor qRT-PCR work?

Answer 13

tagman probe bind to sequence, gets altered when polymerization reaches it –fluorescene changes

Question 14

How does CYBR-Green qRT-PCR work?

Answer 14

sybr green is a dye than caan intercalate into dann and fluoresces upon light excitation when bound to double stranded dann

sample becomes more fluorescent during extension

Question 15

How can qrtpcr be quantified (absolute)?

Answer 15

use of a Dilution series with known number of copiesto create a Standard curve on which the dna Content can be read with the ct value

Question 16

Which 2 qualifiers are important when chocing which sequencing technique to be used?

Answer 16

read length

reads/run

Question 17

What is deep sequencing? (NGS)

Answer 17

sequencing of genomes of a Population of cells

see how populations evolve, see variants arise and compete, to study metagenomes (gut soil ect)

Question 18

How do cDNA microarrays work and what are they used for?

Answer 18

Arrangement of cDNA on microscopy slide,
let cells grow on Glucose or Ethanol medium, Isolate total mrna, reverse transcribe to cdna labeld with a fluorescent dye, mix, hybridize to dann microarray, wash, measure green and red fluorescence over each spot

quantify rRNA Levels

Question 19

How can RNA be sequenced and quantified?

Answer 19

mrna Isolation
sequencing
align sequences against Genome
generate sequence Counts for all genes in Genome
gene A 30/10 = 3 fold Change
gene B 10/5 = 2 fold change

Question 20

4 immuno basedmethods for alaysis

Answer 20

immunofluorescence (prot and cells) (ab recognition sex ab with fluorescent probe

FACS fluorescence assosiated cell sorting
ELISA (prot)
Western blot, recognition of prot on Membrane with prim, sec ab

Question 21

How does 2D Gel electrophoresis work?

Answer 21

1. isoelectric focusing gel -> on sds plyacrylamide gel (sep by mass)

after Separation identification with diff methods, prot sequencing

Question 22

What is mass spectrometry and what is it good for?

Answer 22

measuring the mass of Atoms in a molecule
identification of prot
can be done with diff Labels and tags

Question 23

How can B be identified? (MALDI-TOF MS, pyrosequencing)

Answer 23

malditof uses mass spectromety on bac samples to identify species
compares unknown sample to data of known samples

or Pyrosequencing: Cell culture, Isolate dnaa pcr pyrosequencing with dTTP, programs gets fluorescent Signals for diff nucleotides
identification of species sequence genome